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Abstract Abstract Multiplex PCR technique of microsatellites has been wildly used in the field of parent assignment, population genetic analysis and family management. Rhinogobio ventralis is endemic and rare fish in the upper Yangtze River, China. From the species vale and threatened degreed evaluation, it has reached three-level desperate protection sate and has been classified as low risk fish. In order to develop a rapid, economically efficient and robust approach for genetic studies of R. ventralis, a technique for the parentage assignment in R. ventralis was established based on 5 multiplex PCR panels using previously reported microsatellites loci with high polymorphism. The annealing temperatures, the primer ratio and the concentrations of reaction system were optimized in this technique. With the multiplex PCR tool, the genotyping and the genetic diversity analysis on 136 individuals of R. ventralis were performed using ABI 3730 genetic analyzer and Cervus v.3.0 software. The results showed that the average number of alleles (Na) was 15.600; the average expected heterozygosity (He) was 0.825; the average observed heterozygosity (Ho) was 0.813; the average polymorphism information content (PIC) was 0.801. The parentage assignment was performed on 40 candidate parents and 96 offspring. The combined exclusion probability of the first parent (CE-1P), the second parent (CE-2P), and a parent pair (CE-PP) were shown to be 0.999 986 07, 0.999 999 97 and 1.000 000 00, respectively. When using multiplex PCR group A, B and C, the CE-1P was 0.998 348 13 and CE-2P was 0.999 960 49. All of offspring could correctly find their real parents. The accuracy rate of parentage assignment was 100% using 5 multiplex PCR panels. These results indicated that 15 polymorphic loci and microsatellite multiplex PCR technique were suitable for paternity test of R. ventralis. The development of microsatellite multiplex PCR system provides a high efficiency technical method for parentage assignment, genetic diversity analysis, and marker-assisted family management in R. ventralis.
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Received: 14 February 2017
Published: 06 August 2017
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